Protein fragments for use in protein targeting

ABSTRACT

A protein is described. The protein comprises a lipid globule targeting sequence linked to a protein of interest (POI) wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or fragment or homologue thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part application of U.S. patentapplication Ser. No. 09/203,649, filed Dec. 1, 1998 now U.S. Pat. No.6,340,577 and, which claims priority under 35 U.S.C. §119 to GreatBritain patent application No. 9825953.4, filed Nov. 26, 1998.

FIELD OF THE INVENTION

This invention relates to the use of polypeptides derivable from thecore protein of the hepatitis C virus for targeting proteins of interestto lipid globules, in particular lipid globules subsequently secretedinto animal milk. The resulting protein/lipid complexes may be used intherapy including the production of vaccines.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a major causative agent of chronic hepatitisand liver disease. It is estimated that, worldwide, approximately 300million individuals are infected with the virus, 20% of whom are likelyto develop mild to severe liver disease or carcinoma. Apart from therisk of succumbing to the long term effects of infection, theseindividuals also represent a large reservoir of virus for futuretransmissions. To date, the only widely used therapy for HCV istreatment with interferon. However, sustained response is achieved inonly about 20% of cases. Moreover, no vaccine currently exists toprotect against infection. Since growth of the virus has not beenpossible to date in tissue culture systems, very little is known alsoabout the molecular events which occur during viral replication.

The core protein of HCV is predicted to constitute the capsid of virusparticles. From various studies, expression of this protein results in arange of effects on intracellular processes, including a decrease intranscription of genes from HBV and HIV and alterations to apoptosis.There is also evidence from a study on transgenic mice thatliver-specific expression of core may be linked to the development ofsteatosis (fatty liver), a condition commonly found in HCV-infectedindividuals which is characterized by the accumulation of fat depositswithin hepatocytes. Thus, core protein may also influence lipidmetabolism within the liver. Other results from studies on human serasuggest that HCV virus particles are found associated with lipoproteinparticles which are produced by the liver. It has also been shown thatHCV core protein associates with lipid droplets within cells (Barba, G.et al., 1997; Moradpour, D. et al., 1996). The droplets are storagecompartments for both triacylglycerols and cholesterol esters which canbe used as substrates for oxidation in mitochondria and for theformation of membranes. In specialized cells, stored cholesterol is usedfor steroid hormone synthesis.

Within the liver, lipid droplets also function as a site for storage ofprecursors of the lipid which is secreted from this organ in the form oflipoprotein particles. Although lipid droplets were identified severaldecades ago and they can be readily detected by staining methods, verylittle is known about the processes of assembly, storage and disassemblywithin the cell. One protein, termed adipocyte-related differentiationprotein (ADRP), has been found to associate with lipid droplets in arange of cell types and in certain organs. To date, it is the onlyprotein which is apparently not cell-type specific that has thisintracellular distribution. It is proposed that ADRP may be required formaintenance of lipid droplets within cells, however the precise functionof the protein has not been identified.

SUMMARY OF THE INVENTION

Particular sequences within the hepatitis C virus core protein thatdirect association of HCV core protein with intracellular lipid globuleshave now been characterized. These sequences can thus be used to targetother proteins to lipid globules, including lipid globules secreted bymilk-producing cells. We have also shown that expression of core proteinand its resultant association with lipid droplets results in loss ofADRP from the droplets. Furthermore, progressive increases in coreexpression result in diminishing amounts of ADRP to undetectable levels.Since it has been shown previously that ADRP is also secreted as acomponent of fat globules in milk from humans, cows and rats, proteinscomprising HCV core protein sequences may also be secreted into animalmilk. Thus fusion proteins comprising HCV core protein elements fused toproteins of interest may be targeted specifically to lipid globulessecreted into the milk produced by a variety of animals and the proteinsextracted from the milk. This will facilitate the expression andsecretion into milk of proteins of interest and provide an effectivemethod of producing recombinant proteins in transgenic animals.

Accordingly, the present invention provides a protein comprising a lipidglobule targeting sequence linked to a protein of interest (POI) whereinthe targeting sequence comprises a hepatitis C virus (HCV) core proteinor fragment or homologue thereof. Preferably, the lipid globuletargeting sequence comprises amino acids from 125 to 144 and/or 161 to166 of the HCV core protein as set out in SEQ ID. Nos. 2 and 3, or theequivalent amino acids in other HCV strains/isolates. More preferably,the lipid globule targeting sequence also comprises a hydrophilic aminoacid sequence of at least 8 amino acids. The present invention alsoprovides an isolated polypeptide consisting essentially of a lipidglobule targeting sequence wherein the targeting sequence comprises fromamino acids 125 to 144 and 161 to 166 of an HCV core protein linked to ahydrophilic amino acid sequence of at least 8 amino acids.

The protein of interest is preferably a protein expressed by a pathogen,preferably a viral or bacterial protein or fragment thereof, morepreferably comprising at least one epitope.

In another aspect, the present invention provides a polynucleotideencoding a protein of the invention. The present invention also providesa polynucleotide encoding a protein of the invention operably linked toa control sequence permitting expression of the protein in a suitablehost cell. Preferred host cells include adipocytes and milk-secretingcells.

The invention further provides a nucleic acid vector comprising apolynucleotide of the invention. The invention also provides a host cellcomprising a polynucleotide of the invention or a nucleic acid vector ofthe invention.

In another aspect, the present invention provides a method for producinga protein of the invention which method comprises culturing a host cellof the invention under conditions which allow expression of the protein,and recovering the protein.

The proteins of the invention may advantageously be extracted from cellsassociated with the lipid globules to which the proteins have beendirected by the lipid globule targeting sequence. In particular,proteins produced in milk-secreting cells in milk-producing animals mayconveniently be extracted from the animal's milk. These protein/lipidcomplexes may be used without further purification. Indeed, lipids havebeen used as adjuvants in the preparation of vaccine compositions.Consequently, protein/lipid globule compositions of the invention may beused in the preparation of vaccines, in particular where the protein ofinterest is immunogenic.

Thus, the invention also provides a composition comprising a protein ofthe invention and a lipid globule. Preferably the lipid globule is aconstituent of mammalian milk.

The compositions, proteins, polynucleotides and vectors of the presentinvention may be used in the prevention or treatment of pathogenicinfections. Thus, in a further aspect, the present invention provides avaccine composition comprising a composition, protein, polynucleotide orvector of the invention together with a pharmaceutically acceptablecarrier or diluent. It may be preferred to use the proteins of theinvention in combination with the active constituents of other vaccinecompositions to increase their effectiveness.

The present invention also provides a method of treating or preventing apathogenic infection in a human or animal which comprises administeringto the human or animal an amount of a composition, protein,polynucleotide or vector of the invention sufficient to achieve abeneficial immunological effect.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al.,Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

A. Proteins/Polypeptides

The term “protein” includes single-chain polypeptide molecules as wellas multiple-polypeptide complexes where individual constituentpolypeptides are linked by covalent or non-covalent means. The term“polypeptide” includes peptides of two or more amino acids in length,typically having more than 5, 10 or 20 amino acids. Proteins of theinvention generally comprise at least two components—a lipid globuletargeting sequence which is capable of targeting molecules to lipidglobules and a molecule of interest, typically a protein.

1. Lipid Globule Targeting Sequences

The term “lipid globule targeting sequence” means an amino acid sequencewhich is capable of association with a lipid globule, preferably abiologically occurring lipid globule such as an intracellular lipidglobule as found in adipocytes or a secreted lipid globule as found inmammalian milk. In addition, the lipid globule targeting sequence ispreferably capable of association with a lipid globule when linked to aprotein of interest such that the protein of interest is also associatedwith the lipid globule by virtue of being linked to the targetingsequence. Lipid globule association may take place within a non-cellularand/or extra-cellular environment, such as in an apparatus—for example atube or vat. Alternatively, it may take place in a cellular environmentwhere the expressed targeting sequence is directed to intracellularlipid droplets or the membranes of such droplets. It is especiallypreferred that the targeting sequence is directed to lipid dropletswhich are subsequently secreted into the extracellular environment, forexample during the production by female animals of milk.

The ability of an amino acid sequence to associate with/target lipidglobules can be assessed either in vitro or in vivo. For example, acandidate targeting sequence may be added to a dispersion of lipidglobules (such as a mixture of phospholipid and triacylglycerol) in anaqueous solvent, the mixture sonicated and the degree of partitionbetween aqueous and lipid phases determined by fractionation. Typicallyfractionation of the mixture would involve increasing the density of thesolution with sorbitol or sodium bromide and ultracentrifuging thesolution. The lipid complexes migrate to the top of the centrifuge tubeand this upper lipid layer is then examined for candidate targetingsequence. Preferably, a suitable lipid globule targeting sequence shouldpartition at least 50:50 lipid:aqueous phase, more preferably at least75:25, 80:20 or 90:10.

Another suitable test may involve introducing a polynucleotide encodinga candidate sequence, optionally linked to a protein of interest, into amilk-producing cell in culture and determining whether, the targetingsequence/protein of interest has been secreted into the culture medium.The immunocytochemical technique illustrated in the Examples may also beused.

Suitable lipid globule targeting sequences may be obtained from an HCVcore protein.

The amino acid sequence of the HCV core protein has been obtained for alarge number of different HCV isolates. These sequences are readilyavailable to the skilled person. One such sequence, for HCV strainGlasgow, is set out in SEQ ID No. 1. The means for cloning andidentifying new HCV strains, and thus obtaining further core sequences,are described in EP-B-318,216

According to the present invention, it is preferred to use fragments ofthe HCV core protein which are capable of targeting molecules, to whichthey are linked, to lipid globules. Amino acid numbering for preferredfragments set out below is with reference to SEQ ID. No. 1. However itwill be understood that equivalent fragments of the core protein ofother HCV strains/isolates may also be used. An HCV coreprotein-derivable lipid globule targeting sequence of the invention ispreferably a minimal amino acid sequence which can target a molecule,typically a protein, to lipid globules. The minimal sequence willtypically comprise a hydrophobic amino acid sequence derived from aminoacids 120 to 169 of an HCV core sequence, preferably linked to ahydrophilic amino acid sequence of at least 8, preferably 10, morepreferably at least 12 amino acids. It is not necessary for thehydrophilic sequence to be contiguous with the hydrophobic sequence. Forexample, a protein of interest may be placed between the two sequencessuch that the hydrophilic sequence is at the N-terminus and thehydrophobic sequence is at the C-terminus.

The hydrophobic amino acid sequence typically comprises at least 10,preferably at least 15 or 20 contiguous amino acids and has a hydropathyindex of at least +40 kJ/mol (determined, for example, theoretically asdescribed by Engelman et al., 1986). The hydrophilic amino acid sequencetypically has a hydropathy plot of less than −20 kJ/mol, preferably lessthan −40 kJ/mol.

Preferred HCV core fragments contain amino acids 161 to 166 (SEQ ID. No.3). It is also preferred to use fragments of the HCV core protein thatcontain amino acids 125 to 144 (SEQ ID. No. 2). In a preferredembodiment, HCV core protein fragments of the invention contain bothamino acids 125 to 144 and amino acids 161 to 166. In an especiallypreferred embodiment, the lipid targeting sequence of the inventioncomprises a hydrophilic amino acid sequence containing amino acids 1 to8 of the HCV core sequence. Other preferred fragments contain aminoacids 1 to 173 or 1 to 169.

Since it has also now been shown that amino acids 9 to 43, 49 to 75, 80to 118 and 155 to 161 are not required for lipid association, preferredHCV core protein fragments of the invention lack one or more of thesesequences. In particular, it is preferred that HCV core proteinfragments of the invention lack amino acids 9 to 43. Suitable fragmentswill be at least about 5, e.g. 10, 12, 15 or 20 amino acids in size andpreferably have less than 100, 90, 80, 70, 60 or 50 amino acids. In apreferred aspect, fragments contain an HCV epitope.

Lipid globule targeting sequences of the invention, for example HCV coreprotein sequences and fragments thereof, may, however, be part of alarger polypeptide, for example a fusion protein. In this case, theadditional polypeptide sequences are preferably polypeptide sequenceswith which the lipid globule targeting sequence of the invention is notnormally associated.

It will be understood that lipid globule targeting sequences of theinvention are not limited to sequences obtained from HCV core proteinbut also include homologous sequences obtained from any source, forexample related viral proteins, cellular homologues and syntheticpeptides, as well as variants or derivatives thereof. Thus, the presentinvention covers variants, homologues or derivatives of the targetingsequences of the present invention, as well as variants, homologues orderivatives of the nucleotide sequence coding for the targetingsequences of the present invention.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least 5, preferably 8, 10, 15, 20, 30 or 40 amino acidswith an HCV core protein lipid targeting sequence, for example as shownin the sequence listing herein. In particular, homology should typicallybe considered with respect to those regions of the targeting sequenceknown to be essential for lipid globule association rather thannon-essential neighboring sequences. Homology comparisons can beconducted by eye, or more usually, with the aid of readily availablesequence comparison programs. These commercially available computerprograms can calculate % homology between two or more sequences. Atypical example of such a computer program is CLUSTAL.

Sequence homology (or identity) may moreover be determined using anysuitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andare advantageously set to the defined default parameters.

Advantageously, “substantial homology” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (seehttp://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.The BLAST programs were tailored for sequence similarity searching, forexample to identify homologues to a query sequence. The programs are notgenerally useful for motif-style searching. For a discussion of basicissues in similarity searching of sequence databases, see Altschul etal. (1994).

The five BLAST programs available at http://www.ncbi.nlm.nih.gov performthe following tasks:

blastp—compares an amino acid query sequence against a protein sequencedatabase;

blastn—compares a nucleotide query sequence against a nucleotidesequence database;

blastx—compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase;

tblastn—compares a protein query sequence against a nucleotide sequencedatabase dynamically translated in all six reading frames (bothstrands).

tblastx—compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM—Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS—Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page). See also EXPECT andCUTOFF.

ALIGNMENTS—Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

EXPECT—The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF—Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

MATRIX—Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).The valid alternative choices include: PAM40, PAM120, PAM250 andIDENTITY. No alternate scoring matrices are available for BLASTN;specifying the MATRIX directive in BLASTN requests returns an errorresponse.

STRAND—Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993), or segments consisting of short-periodicity internalrepeats, as determined by the XNU program of Claverie & States (1993),or, for BLASTN, by the DUST program of Tatusov and Lipman (seehttp://www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g. hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g., “XXXXXXXXX”).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.

Other computer program methods to determine identify and similaritybetween the two sequences include but are not limited to the GCG programpackage (Devereux et al., 1984) and FASTA (Atschul et al., 1990).

Lipid globule targeting sequences of the invention, for example HCV coreprotein sequences, variants, homologues and fragments thereof, may bemodified for use in the present invention. Typically, modifications aremade that maintain the hydrophobicity/hydrophilicity of the sequenceAmino acid substitutions may be made, for example from 1, 2 or 3 to 10,20 or 30 substitutions provided that the modified sequence retains theability to target molecules to lipid globules. Amino acid substitutionsmay include the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other.

The terms “variant”, “homologue” or “derivative” in relation to thetargeting sequence of the present invention include any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has a lipid globule targeting activity,preferably having at least the same activity of the targeting sequencepresented in the sequence listings.

ALIPHATIC Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DEKR AROMATIC HFWY

The terms “variant”, “homologue” or “derivative” in relation to thetargeting sequence of the present invention include any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has a lipid globule targeting activity,preferably having at least the same activity of the targeting sequencepresented in the sequence listings.

2. Proteins of Interest

Proteins of interest may include, for example, proteins involved in theregulation of cell division, for example growth factors includingneurotrophic growth factors, cytokines (such as α-, β- or γ-interferon,interleukins including IL-1, IL-2, tumor necrosis factor, orinsulin-like growth factors I or II), protein kinases (such as MAPkinase), protein phosphatases and cellular receptors for any of theabove. The protein may also be an enzyme involved in cellular metabolicpathways, for example enzymes involved in amino acid biosynthesis ordegradation (such as tyrosine hydroxylase), purine or pyrimidinebiosynthesis or degradation, and the biosynthesis or degradation ofneurotransmitters, such as dopamine, or a protein involved in theregulation of such pathways, for example protein kinases andphosphatases. The protein may also be a transcription factors orproteins involved in their regulation, for example pocket proteins ofthe Rb family such as Rb or p107, membrane proteins, structural proteinsor heat shock proteins such as hsp70. Proteins of interest arepreferably lipid soluble or contain regions which allow a portion of theprotein to be buried in a lipid globule. Preferably the POI will nothinder the lipid targeting effect of the lipid globule targetingsequence.

Preferably, the protein of interest is of therapeutic use, or thefunction of which may be implicated in a disease process. Proteins ofinterest may also contain antigenic polypeptides for use as vaccines.Preferably such antigenic polypeptides are derived from pathogenicorganisms, for example bacteria or viruses, or from tumors. Inparticular antigenic polypeptides containing HCV epitopes may be used.Extensive epitope mapping of the HCV genome has already been carried outand the majority of HCV epitopes characterized. Epitopes may be linearor conformational. In the case of HCV core protein epitopes, the HCVcore protein targeting sequence of the invention may already containsuitable HCV epitopes and this being the case, it may not be necessaryto include further antigenic sequences. Consequently an HCV core proteinsequence may be used according to the present invention without beingfused to a protein of interest. However, proteins of interest shouldpreferably not be sequences with which the lipid globule targetingsequences are normally associated.

In addition to being linked to the lipid globule targeting sequence,proteins of interest may be linked to further fusion proteins.Polypeptides of the invention may also be produced as fusion proteins,for example to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6×His, GAL4(DNA binding and/or transcriptional activation domains) andβ-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the HCV coreprotein sequence and/or between the HCV core protein sequence and theprotein of interest to allow removal of fusion protein sequences.Preferably the fusion protein will not hinder the lipid targeting effectof the lipid globule targeting sequence. The targeting sequence may belinked to either the N-terminus or the C-terminus of the fusion proteinpartners or proteins of interest

Proteins of the invention are typically made by recombinant means, forexample as described below. However they may also be made by syntheticmeans using techniques well known to skilled persons such as solid phasesynthesis.

Proteins of the invention may be in a substantially isolated form. Itwill be understood that the protein may be mixed with carriers ordiluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A protein ofthe invention may also be in a substantially purified form, in whichcase it will generally comprise the protein in a preparation in whichmore than 90%, e.g. 95%, 98% or 99% of the protein in the preparation isa protein of the invention.

B. Polynucleotides and Vectors.

Polynucleotides of the invention comprise nucleic acid sequencesencoding the lipid globule targeting sequences of the invention andproteins of the invention. It will be understood by a skilled personthat numerous different polynucleotides can encode the same polypeptideas a result of the degeneracy of the genetic code. In addition, it is tobe understood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides of the invention to reflect the codonusage of any particular host organism in which the polypeptides of theinvention are to be expressed.

Polynucleotides of the invention may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span of polynucleotidesof the invention.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence coding for the lipid targeting sequence of thepresent invention include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for a protein having lipid targeting activity,preferably having at least the same activity of the targeting sequencepresented in the sequence listings.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology to the sequences shown in the sequence listing herein. Morepreferably there is at least 95%, more preferably at least 98%,homology. Nucleotide homology comparisons may be conducted as describedabove.

The present invention also encompasses nucleotide sequences that arecapable of hybridizing selectively to the sequences presented herein, orany variant, fragment or derivative thereof, or to the complement of anyof the above. Nucleotide sequences are preferably at least 15nucleotides in length, more preferably at least 20, 30, 40 or 50nucleotides in length.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.) as well as the process of amplification as carriedout in polymerase chain reaction technologies as described inDieffenbach C W and G S Dveksler (1995, PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview N.Y.).

Polynucleotides of the invention capable of selectively hybridizing tothe nucleotide sequences presented herein, or to their complement, willbe generally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequence presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides. Preferred polynucleotides of the inventionwill comprise regions homologous to nucleotides 715 to 774 and/ornucleotides 826 to 840 of SEQ ID No. 1, preferably at least 80 or 90%and more preferably at least 95% homologous to to nucleotides 715 to 774and/or nucleotides 826 to 840 of SEQ ID No. 1.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide of theinvention is found to hybridize to the probe at a level significantlyabove background. The background hybridization may occur because ofother polynucleotides present, for example, in the cDNA or genomic DNAlibrary being screened. In this event, background implies a level ofsignal generated by interaction between the probe and a non-specific DNAmember of the library which is less than 10 fold, preferably less than100 fold as intense as the specific interaction observed with the targetDNA. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm−5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridize to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ citrate pH 7.0).

Where the polynucleotide of the invention is double-stranded, bothstrands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other HCV core protein variants of the HCVcore protein sequence described herein may be obtained for example byprobing DNA libraries made from a range of HCV infected individuals, forexample individuals from different populations. In addition, otherviral, or cellular homologues particularly cellular homologues found inmammalian cells (e.g. rat, mouse, bovine and primate cells), may beobtained and such homologues and fragments thereof in general will becapable of selectively hybridizing to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of SEQ ID.1 under conditions of medium to high stringency.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the lipid globule targeting sequences of the presentinvention. Conserved sequences can be predicted, for example, byaligning the HCV core protein amino acid sequences from several HCVisolates. Such HCV sequence comparisons are widely available in the art.The primers will contain one or more degenerate positions and will beused at stringency conditions lower than those used for cloningsequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterized lipid globule targeting sequences, such asSEQ ID. No 1. This may be useful where for example silent codon changesare required to sequences to optimize codon preferences for a particularhost cell in which the polynucleotide sequences are being expressed.Other sequence changes may be desired in order to introduce restrictionenzyme recognition sites, or to alter the property or function of thepolypeptides encoded by the polynucleotides.

Polynucleotides of the invention may be used to produce a primer, e.g. aPCR primer, a primer for an alternative amplification reaction, a probee.g. labeled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein.

Polynucleotides such as a DNA polynucleotides and probes according tothe invention may be produced recombinantly, synthetically, or by anymeans available to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the lipid targeting sequence/POIwhich it is desired to clone, bringing the primers into contact withmRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell.

Thus in a further embodiment, the invention provides a method of makingpolynucleotides of the invention by introducing a polynucleotide of theinvention into a replicable vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector. The vector may be recovered fromthe host cell. Suitable host cells include bacteria such as E. coli,yeast, mammalian cell lines and other eukaryotic cell lines, for exampleinsect Sf9 cells.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence that is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

Such vectors may be transformed or transfected into a suitable host cellas described below to provide for expression of a protein of theinvention. This process may comprise culturing a host cell transformedwith an expression vector as described above under conditions to providefor expression by the vector of a coding sequence encoding the protein,and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell either in vitro or invivo.

Control sequences operably linked to sequences encoding the protein ofthe invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell for which the expression vector isdesigned to be used in. The term promoter is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

The promoter is typically selected from promoters which are functionalin mammalian, cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression of the protein is to occur. With respect to eukaryoticpromoters, they may be promoters that function in a ubiquitous manner(such as promoters of α-actin, β-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of the genes for pyruvatekinase). Tissue-specific promoters specific for adipocyte cells (such asthe perilipin promoter), in particular milk-producing cells, areparticularly preferred, for example promoters for α-lactalbumin,β-lactoglobulin, whey acidic protein or butyrophilin genes. They mayalso be promoters that respond to specific stimuli, for examplepromoters that bind steroid hormone receptors. Viral promoters may alsobe used, for example the Moloney murine leukaemia virus long terminalrepeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter orthe human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the POI can be regulated during thelife-time of the cell. Inducible means that the levels of expressionobtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

C. Host Cells

Vectors and polynucleotides of the invention may be introduced into hostcells for the purpose of replicating the vectors/polynucleotides and/orexpressing the proteins of the invention encoded by the polynucleotidesof the invention. Although the proteins of the invention may be producedusing prokaryotic cells as host cells, it is preferred to use eukaryoticcells, for example plant, yeast, insect or mammalian cells, inparticular mammalian cells. Particularly preferred cells are those withsubstantial amounts of intracellular lipid droplets/globules, forexample adipocytes. In a preferred embodiment, host cells which secretelipid globules, for example milk-producing cells, are used. Mammaliancell lines may be transfected in vitro or alternatively, intactmulticellular organisms may be used, for example ungulates such as cows,goats, pigs and sheep. Preferably animals with high milk yields areused.

Vectors/polynucleotides of the invention may be introduced into suitablehost cells using a variety of techniques known in the art, such astransfection, transformation and electroporation. Wherevectors/polynucleotides of the invention are to be administered toanimals, several techniques are known in the art, for example infectionwith recombinant viral vectors such as herpes simplex viruses andadenoviruses, direct injection of nucleic acids and biolistictransformation. Alternatively, transgenic animals may be produced usingsuitable techniques.

For example, one method used to produce a transgenic animal involvesmicroinjecting a nucleic acid into pro-nuclear stage eggs by standardmethods. Injected eggs are then cultured before transfer into theoviducts of pseudopregnant recipients. Analysis of animals which maycontain transgenic sequences would be performed by either PCR orSouthern blot analysis following standard methods.

Transgenic animals may also be produced by nuclear transfer technologyas described in Schnieke, A. E. et al. (1997) and Cibelli, J. B. et al.(1998). Using this method, fibroblasts from donor animals are stablytransfected with a plasmid incorporating the coding sequences for coreor any proteins of interest fused to lipid globule targeting sequencesunder the control of regulatory elements required for optimal expressionin mammary cells. Stable transfectants are then fused to enucleatedoocytes, cultured and transferred into female recipients.

When constructing suitable nucleic acids of the invention forintroduction into mammalian eggs during production of transgenicanimals, regulatory sequences typically used are promoter elements thatare required for tissue-specific expression, examples of which arelisted in Section B. Additionally, regulatory sequences may includeintrons, enhancer elements and sequences flanking the portion of thecoding region which are known to influence expression in transgenicanimals and may be required for optimal expression in milk. Theseregulatory elements may be of natural or synthetic origin and placedupstream of, within and downstream of the coding sequences. The nucleicacid vector used for production of transgenic animals may incorporatealso the entire β-lactoglubulin gene. Such methodology is known toincrease expression levels in transgenic animals (see for example Sola,I. et al., 1998).

D. Protein Expression and Purification

Host cells comprising polynucleotides of the invention may be used toexpress proteins of the invention. Host cells may be cultured undersuitable conditions which allow expression of the proteins of theinvention. Expression of the proteins of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, protein production can be initiated when required by, forexample, addition of an inducer substance to the culture medium, forexample dexamethasone or IPTG.

Proteins of the invention can be extracted from host cells by a varietyof techniques known in the art, including enzymatic, chemical and/orosmotic lysis and physical disruption. Although a large number ofdifferent purification protocols may be used, given the ability of theHCV core proteins of the invention to target proteins of interest tolipid globules, a preferred extraction/purification protocol involvescentrifuging cell homogenates at high speed (for example 100, 000 g for60 mins at 2 to 4° C.) and removing the resulting layer of floatinglipids. This will function as a primary purification step. Furtherpurification can then be performed if necessary using, for example,column chromatography such as ion-exchange or affinity chromatography.Cells which secrete lipid globules may also conveniently be used and thelipid globules harvested from the culture supernatant.

Proteins associated with the membrane surrounding fat globules can befractionated into soluble and insoluble fractions by extraction with 1%(w/v) Triton X-100/1.5 M NaCl/10 mM Tris (pH 7.0), by extraction with1.5% (w/v) dodecyl β-D maltoside/0.75 M aminohexanoic acid/10 mM Hepes(pH 7.0) or by sequential extraction with these two detergent-containingsolutions (Patton, S. and Huston, G. E., 1986, Lipids 21; 170-174).Suspension of the fat globule components in the detergent-containingsolution can be achieved by using an all-glass homogenizer, and keepingon ice for 30 to 60 min, after which insoluble and soluble materials canbe separated by centrifugation for 60 min at 2° C. and 150,000 g. Theabove conditions can be modified to analyse whether core protein or afusion protein containing core as a component is attached to fatglobules. Other detergents, both ionic and non-ionic, along with saltsolutions at various concentrations could be used to derive theproteinaceous material from fat globules. The incubation times andtemperatures may be optimized by empirical means.

A particularly preferred method for producing proteins of the inventioninvolves using milk-producing animals stably transfected with suitableexpression vectors, or transgenic milk-producing animals. In thesecases, the milk is harvested from the animals, and the lipidglobule/protein complex extracted.

Milk fat globules can be separated from whole milk by centrifugation at2000 g for 15 min at room temperature where they collect as a layer atthe top of the centrifuge tube (Freudenstein, C. et al., 1979).Alternatively, sucrose can be added to milk (5% w/v) and this milksolution can be layered below an overlying layer of water, buffer orsaline solution. Following centrifugation at 2000 g for 20 min at roomtemperature, milk fat globules collect as a layer at the top of thecentrifuge tube. In both methods, fat globules can be collected by aspoon, pipette or similar device. To enhance purity, the fat globulescan be dispersed in a saline solution and collected by centrifugation asdescribed above. These methods are suitable for volumes of less than 1ml up to approximately 1 liter. For greater volumes, a cream separatorcould be employed.

E. Compositions

Proteins of the invention may be combined with various components toproduce compositions of the invention. These components may includepharmaceutically acceptable carriers or diluents, and/or vaccinecomponents as described below. In particular, a composition of theinvention comprises a protein of the invention together with a lipidglobule. Since the HCV core protein of the invention targets proteins ofinterest to lipid globules, one of the products of the purificationprocedure may be the protein of interest already associated with a lipidglobule. Alternatively, proteins of the invention may be produced and/orextracted to provide an aqueous product, substantially free ofassociated lipids, and lipid globules added to the purified product.Preferred lipid globules are those which occur in mammalian milk.

F. Administration

The compositions of the invention may be administered by directinjection. Preferably the compositions are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition (which may be for human or animal use).Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The composition may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular ortransdermal administration. Typically, each protein may be administeredat a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

The polynucleotides/vectors of the invention may be administereddirectly as a naked nucleic acid construct, preferably furthercomprising flanking sequences homologous to the host cell genome. Whenthe polynucleotides/vectors are administered as a naked nucleic acid,the amount of nucleic acid administered is typically in the range offrom 1 μg to 10 mg, preferably from 100 μg to 1 mg.

Uptake of naked nucleic acid constructs by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Preferably the polynucleotide or vector of the invention is combinedwith a pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular or transdermal administration.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage for any particularpatient and condition.

G. Preparation of Vaccines

Vaccines may be prepared from one or more proteins of the invention orcompositions of the invention where the proteins are immunogenic, forexample comprising epitopes from viral or bacterial pathogens. They mayalso include one or more additional immunogenic polypeptides known inthe art. The preparation of vaccines which contain an immunogenicpolypeptide(s) as active ingredient(s), is known to one skilled in theart. Typically, such vaccines are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the protein encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against an immunogenicpolypeptide containing an antigenic sequence resulting fromadministration of this polypeptide in vaccines which are also comprisedof the various adjuvants.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and may contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilized, the lyophilized material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer.

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose. These capsules may be used as such, oralternatively, the proteins and compositions of the invention may beformulated into the vaccine as neutral or salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with free aminogroups of the peptide) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or such organic acidssuch as acetic, oxalic, tartaric and maleic. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine.

H. Dosage and Administration of Vaccines

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective. The quantity to be administered, which maygenerally be in the range of 5 mg to 250 mg of antigen per dose, dependson the subject to be treated, capacity of the subject's immune system tosynthesize antibodies, and the degree of protection desired. Preciseamounts of active ingredient required to be administered may depend onthe judgement of the practitioner and may be peculiar to each subject.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1 to 10 separate doses,followed by other doses given at subsequent time intervals required tomaintain and or reinforce the immune response, for example, at 1 to 4months for a second dose, and if needed, a subsequent dose(s) afterseveral months. The dosage regimen will also, at least in part, bedetermined by the need of the individual and be dependent upon thejudgement of the practitioner.

In addition, the vaccine containing the immunogenic proteins of theinvention may be administered in conjunction with other immunoregulatoryagents, for example, immunoglobulins.

I. Preparation of Antibodies Against the Polypeptides of the Invention

The immunogenic proteins of the invention prepared as described abovecan be used to produce antibodies, both polyclonal and monoclonal. Ifpolyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunized with an immunogenic protein ofthe invention. Serum from the immunized animal is collected and treatedaccording to known procedures. If serum containing polyclonal antibodiesto an immunogenic protein of the invention contains antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art.

Monoclonal antibodies directed against epitopes of interest in theproteins of the invention can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal antibody-producing cell lines can becreated by cell fusion, and also by other techniques such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus. Panels of monoclonal antibodies produced againstepitopes of interest can be screened for various properties; i.e., forisotype and epitope affinity.

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes are particularly useful in diagnosis, and those which areneutralizing are useful in passive immunotherapy. Monoclonal antibodies,in particular, may be used to raise anti-idiotype antibodies.Anti-idiotype antibodies are immunoglobulins which carry an “internalimage” of the antigen of the infectious agent against which protectionis desired.

Techniques for raising anti-idiotype antibodies are known in the art.These anti-idiotype antibodies may also be useful for treatment of viraland/or bacterial diseases, as well as for an elucidation of theimmunogenic regions of viral and/or bacterial antigens.

It is also possible to use fragments of the antibodies described above,for example, Fab fragments.

The invention will be described with reference to the following Exampleswhich are intended to be illustrative only and not limiting. TheExamples refer to the following Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Western blots probed with antibodies to HCV core protein

FIG. 2 shows confocal microscopy images of the intracellularlocalization of core proteins and lipid droplets.

FIG. 3 shows confocal microscopy images of cells illustrating the effectof expression of HCV core proteins on the ability to detect ADRP inBHKC13 cells.

FIG. 4 shows confocal microscopy images of cells illustrating the effectof expression of HCV core protein on the abundance of ADRP.

FIG. 5 shows Western blots probed with antibodies to HCV core proteinand adipophilin.

FIG. 6 shows the amino acid sequence comparison between the predictedcore proteins of HCV and GBV-B.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1

Analysis of the core proteins made by the pSFV and pgHCV constructs.

A. Western blot analysis of extracts prepared from cells which wereharvested 20 hours after electroporation. Aliquots of extractscontaining the same number of cell equivalents were analyzed withantibody JM122. The samples were from cells electroporated with RNA fromthe following constructs: lane 1, pSFV.1-195; lane 2, pSFV.1-173; lane3, pSFV.1-169; lane 4, pSFV.1-153; lane 5, pSFV.Δ155-161; lane 6, pSFV.Δ161-166; lane 7, no RNA.

Arrows denote the forms of core which have (labeled C) and have not(labeled UC) been cleaved at the internal processing site.

B. In vitro translation of core proteins. Products of reactions wereelectrophoresed on a 10% polyacrylamide gel and detected byautoradiography. The samples were from reactions containing thefollowing constructs: lane 1, pgHCV.1-195; lane 2, pgHCV.1-173; lane 3,pgHCV.1-153.

FIG. 2

Confocal images of the intracellular localization of core proteins andlipid droplets. BHK C13 cells were harvested 20 hours afterelectroporation and fixed with 4% paraformaldehyde, 0.1% Triton X-100.Indirect immunofluorescence was performed with antibody JM122 and ananti-mouse secondary antibody conjugated with FITC. Lipid droplets werestained with oil red O. Panels A, D, G, J, M, P, S and V show thedistributions of core protein. Panels B, E, H, K, N, Q, T and W show thelocations of lipid droplets. Panels C, F, I, L, O, R, U and X are mergedimages of core protein and lipid droplets. Cells were electroporatedwith RNA from the following constructs: panels A, B and C, pSFV.1-195;panels D, E and F, pSFV.1-173; panels G, H and I, pSFV.1-169; panels J,K and L, pSFV.1-153; panels M, N and O, pSFV.Δ155-161; panels P, Q andR, pSFV.Δ161-166; panels S, T and U, pSFVΔ125-144; panels V, W and X,pSFV.1-124, 145-152.

FIG. 3

Effect of expression of core proteins on the ability to detect ADRP inBHKC13 cells by confocal microscopy. Cells were harvested 20 hours afterelectroporation and fixed with methanol. ADRP was detected withanti-adipophilin antibody and core protein with 308 antisera. Secondaryantibodies were an anti-mouse IgG conjugated with FITC (foranti-adipophilin) and anti-rabbit IgG conjugated with Cy5 (for 308antisera). Panels A, D, G, J, M and P are images of ADRP localization.Panels B, E, H, K, N, and Q are images of core distribution. Panels C,F, I, L, O and R show the merged images of core and ADRP distributions.Cells were electroporated with RNA from the following constructs: panelsA, B and C, pSFV.1-195; panels D, E and F, pSFV.1-173; panels G, H andI, pSFV.1-169; panels J, K and L, pSFV.1-153; panels M, N and 0,pSFV.Δ155-161; panels P, Q and R, pSFV.Δ161-166.

FIG. 4

Effect of expression of core proteins on the ability to detect ADRP inMCA RH7777 cells by confocal microscopy. Cells were examined asdescribed in the legend for FIG. 3.

FIG. 5

Effect of expression of core protein on the abundance of ADRP. BHK C13cells were electroporated with RNA from pSFV.1-195 and pSFV.1-153 andextracts were prepared at the times indicated following electroporation.Aliquots of cell extracts were electrophoresed on 10% polyacrylamidegels and then the proteins were transferred to nitrocellulose membranefor Western blot analysis. The upper panels show membranes probed withJM122 antibody while, in the lower panels, membranes were probed withanti-adipophilin antibody. Bands corresponding to core proteins,expressed from pSFV.1-195 and pSFV.1-153, and ADRP are arrowed.

EXAMPLES Materials and Methods

Cell Lines

Baby hamster kidney (BHK) C13 cells were maintained in Glasgow modifiedEagle's medium supplemented with 10% newborn calf serum, 100 IU/mlpenicillin/streptomycin and 5% tryptose phosphate broth. The rathepatoma cell line, MCA RH7777, was maintained in minimal essentialEagle's medium supplemented with 20% foetal bovine serum, 100 IU/mlpenicillin/streptomycin, 1×non-essential amino acids and 2 mML-glutamine.

Immunological Reagents

Antibody JM122 was a mouse monoclonal antibody raised against a purifiedfusion protein, expressed in bacteria, which was composed of theN-terminal 118 amino acid residues of core protein encoded by HCV strainGlasgow linked to a histidine tag. Antisera 308 was raised in rabbitsagainst a branched peptide ([A/P]KPQRKTKRNT[I/N]RRPQDVKFPGG)₈K₇A. Thepeptide consists of residues 5-27 of core protein encoded by HCV strainGlasgow (SEQ ID. No. 1). The two degenerate sites at positions 1 and 12were introduced to obtain antisera which would be reactive against coreproteins from other isolates. The adipophilin antibody was obtained fromCymbus Biotechnology Ltd.

Secondary antibodies were obtained from Sigma with the exception of Cy5conjugated goat anti-rabbit IgG which was obtained from Amersham.

Construction of Plasmids

Plasmids containing the coding region for the core protein of HCV strainGlasgow were obtained by combining fragments from two constructs calledcore.pTZ18 and 5′-ΔNS2 (provided by M. McElwee and R. Elliott).Core.pTZ18 possesses nucleotide residues 337-915 of the HCV strainGlasgow genome and 5′-ΔNS2 contains residues 1-2895. DNA fragments fromthese plasmids were combined in a vector called pGEM1 to give aconstruct termed pgHCV.CE1E2. This plasmid contains nucleotide residues337-2895 of the HCV strain Glasgow genome and therefore encodes thecore, E1 and E2 proteins of this isolate.

For cloning purposes, the sequences immediately upstream of residue 337were modified to contain the recognition sequences for Bgl II and Kpn Irestriction enzyme sites and immediately downstream of residue 2895, anoligonucleotide was inserted which encodes a translational stop codonfollowed by the sequences for a Bgl II restriction enzyme site. Tocreate pgHCV.CE1E2, a Bgl II DNA fragment containing the core, E1 and E2sequences was inserted into the Bam HI site of pGEM1; this plasmid wasfurther modified by introducing a Bgl II enzyme site at the Eco RI sitein the pGEM backbone. Construction of a derivative plasmid, pgHCV.1-195was achieved by inserting an oligonucleotide (GCTGAGATCTA) that had botha translational stop codon and the sequences for a Bgl II enzyme sitebetween a Fsp I enzyme site at residue 925 in the HCV genome and a HindIII enzyme in the pGEM backbone. Thus, pgHCV.1-195 encodes theN-terminal 195 amino acids of HCV strain Glasgow. The nucleotide andpredicted amino acid sequence of this region of HCV strain Glasgow isshown in SEQ ID. No. 1. From pgHCV.1-195, the following series ofconstructs were made which had various regions of the HCV coding regionremoved (in 1 to 3 and 6, the numbers following pgHCV represent theamino acid residues of HCV strain Glasgow encoded by each construct):

1. pgHCV.1-173 was constructed by inserting an oligonucleotideGTAACCTTCCTG GTTGCTCTTGAGATCTA between the Bst EII (at nucleotideresidue 841 in the HCV strain Glasgow genome) and Hind III enzyme sites(located in the pGEM backbone) in pgHCV.1-195.

2. pgHCV.1-169 was constructed by inserting an oligonucleotideGTAACCTTTGAG ATCTA between the Bst EII (at nucleotide residue 841 in theHCV strain Glasgow genome) and Hind III enzyme sites (located in thepGEM backbone) in pgHCV.1-195.

3. pgHCV.1-153 was constructed by inserting an oligonucleotideCTGGCGCATTGA GATCTA between the Bst XI (at nucleotide residue 792 in theHCV strain Glasgow genome) and Hind III enzyme sites (located in thepGEM backbone) in pgHCV.1-195.

4. pgHCV.Δ155-161 was constructed by inserting an oligonucleotideCTGGCCCATG GTGTTAACTATGCAACAG between the Bst XI and Bst EII enzymesites (at nucleotide residues 792 and 841 respectively in the HCV strainGlasgow genome) in pgHCV.1-195. This construct lacks the nucleotidesequences encoding residues 155 to 161 of the core protein of HCV strainGlasgow.

5. pgHCV.Δ161-166 was constructed by inserting another oligonucleotideCTGGCCCATGGCGTCCGGGTTCTGGAAGACG between the Bst XI and Bst EII sites inpgHCV.1-195. This construct lacks the nucleotide sequences encodingresidues 161 to 166 of the core protein of HCV strain Glasgow.

6. pgHCV.1-124, 145-152 was constructed by inserting an oligonucleotideCGATA GAGGCGCTGCCAGGGCCCTGGCGTGAGATCTA between the Cla I (at nucleotideresidue 710 in the HCV strain Glasgow genome) and Hind III enzyme sites(located in the pGEM backbone) in pgHCV.1-195.

7. pgHCV.Δ125-144 was constructed by inserting a 400 bp Kpn I/Bst XI DNAfragment from pgHCV.1-124,145-152 (which contains residues 1-124 and145-152) into a 2970 bp Kpn I/Bst XI DNA fragment from pgHCV.1-195(which contains residues 153-195).

For expression in tissue culture cells, Bgl II DNA fragments carryingthe relevant HCV sequences were prepared from the pgHCV plasmid seriesand inserted into the Bam HI site of a Semliki Forest virus vectorpSFV1. The resultant plasmids were termed the pSFV. series (e.g.pSFV.1-195).

In Vitro Translation

Proteins were translated in vitro using a coupledtranscription/translation kit supplied by Promega. Reactions used 1 μgof DNA as template and were carried out according to manufacturer'sinstructions.

In Vitro Transcription

Prior to electroporation, RNA was transcribed in vitro from theappropriate pSFV plasmid which had been linearised at a Spe I enzymesite. Typical reactions were carried out in a volume of 20 □1 andcontained 40 mM Tris (pH 7.5), 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl,1 mM DTT, 1 mM ATP, 1 mM CTP, 1 mM UTP, 0.5 mM GTP, 1 mM m⁷G(5′)ppp(5′)Gcap analogue, 50 units Rnasin, 50 units SP6 RNA polymerase and 2 μglinearised DNA. Reactions were performed at 37° C. for 2 hours. Productsof the reaction were analyzed by agarose gel electrophoresis to examinethe quality and quantity of RNA synthesized prior to use inelectroporations.

Preparation of Cells Competent for Electroporation

Cells were washed and treated with trypsin for detachment from tissueculture containers. Detached cells were suspended in 20 ml of growthmedium and centrifuged at 100 g for 5 min at room temperature. Cellpellets were suspended in 50 ml of PBSA and centrifuged as previously.Pellets were suspended in PBSA at a final concentration of about 2×10⁷cells/ml.

Electroporation of Cells and Preparation of Cell Extracts

0.8 ml of competent cells were mixed with in vitro transcribed RNA in anelectroporation cuvette (0.4 cm gap) and pulsed twice at either 1.2 kV,25 μF (for BHK C13 cells) or 0.36 kV, 960 μF (for MCA RH7777 cells).Between pulses, the cell/RNA suspension was gently mixed. Followingelectroporation, cells were diluted in growth medium and seeded ontoeither tissue culture dishes or coverslips in 24-well tissue cultureplates and then incubated at 37° C.

To prepare extracts, electroporated cells were harvested by removing thegrowth medium and washing the cell monolayers with PBS. Cells werescraped into PBS and pelleted by centrifugation at 100 g for 5 mins at4° C. The cell pellet was solubilised in sample buffer consisting of 160mM Tris (pH 6.7), 2% SDS, 700 mM β-mercaptoethanol, 10% glycerol, 0.004%bromophenol blue.

Alternatively, sample buffer was added directly to cells which had beenwashed with PBS. Cells were solubilised at a concentration ofapproximately 4×10⁶ cell equivalents per ml sample buffer. Samples wereheated to 100° C. for 5 mins to fully denature proteins and nucleicacids.

SDS-PAGE and Western Blot Analysis

Samples were prepared for electrophoresis and proteins were separated onpolyacrylamide gels cross-linked with 2.5% (wt/wt) N,N′-methylenebisacrylamide using standard techniques. Polypeptides were detectedeither by autoradiography or by staining using Coomassie brilliant blue.

For Western blot analysis, proteins were separated on polyacrylamidegels and transferred to nitrocellulose membrane using standardtechniques. The nitrocellulose membrane was blocked in 3% gelatin, 20 mMTris (pH 7.5), 500 mM NaCl for at least 6 hours at 37° C. prior toincubation with the primary antibody. Incubations with the primaryantibody (diluted to 1/500 for adipophilin antibody and 1/1000 forJM122) were performed in 1% gelatin, 20 mM Tris (pH 7.5), 500 mM NaCl,0.05% Tween 20 at either room temperature or 37° C. for approximately3-4 hours. Following extensive washing with 20 mM Tris (pH 7.5), 500 mMNaCl, 0.05% Tween 20, the membrane was incubated for 2 hours at roomtemperature with anti-mouse IgG conjugated with horse radish peroxidasein the same solution as for the primary antibody and at a dilution of1/1000. Bound antibody was detected by enhanced chemiluminescence.

Indirect Immunofluorescence and Staining of Lipids

Cells on 13 mm coverslips were fixed in either methanol at −20° C. or 4%paraformaldehyde, 0.1% Triton X-100 (prepared in PBS) at 4° C. for 30mins. Following washing with PBS and blocking with PBS/CS (PBScontaining 1% newborn calf serum), cells were incubated with primaryantibody (diluted in PBS/CS at 1/200 for JM122 antibody, 1/1000 for 308antisera and 1/100 for adipophilin antibody) for 2 hours at roomtemperature. Cells were washed extensively with PBS/CS and thenincubated with conjugated secondary antibody (either anti-mouse oranti-rabbit IgG raised in goat) for 2 hours at room temperature. Cellswere washed extensively in solutions of PBS/CS followed by PBS andfinally H₂O before mounting on slides using Citifluor. Samples wereanalyzed using a Leiss LSM confocal microscope.

Following incubation with both antibodies and washing, lipid dropletswere stained in paraformaldehyde-fixed cells by briefly rinsingcoverslips in 60% propan-2-ol followed by incubation with 0.5 ml 60%propan-2-ol containing oil red O for 1.5-2 mins at room temperature.Coverslips were briefly rinsed with 60% propan-2-ol, washed with PBS andH₂O and mounted as described above. The oil red O staining solution wasprepared from a saturated stock of approximately 1% oil red O dissolvedin propan-2-ol. Before staining, the stock was diluted with H₂O and thenfiltered.

Results Example 1 Expression of HCV Core Protein and Variants in TissueCulture Cells

Presently, there is no system available for propagating HCV in tissueculture cells. Therefore, expression of HCV gene products necessitatesthe use of heterologous expression systems. For short-term expression inmammalian cells, a variety of viral vectors have been utilized includingvaccinia virus, Sendai virus and adenovirus. A further alternative isthe Semliki Forest virus (SFV) system in which in vitro transcribed RNA,that encodes the SFV replication proteins as well as a heterologousprotein but not the SFV structural proteins, is introduced into tissueculture cells. Introduction of nucleic acid into cells may be achievedby several routes but, in the examples given, the method of choice iselectroporation.

BHK cells were electroporated with RNA from the series of pSFVconstructs. 20 hours following electroporation, cells were harvested andextracts prepared. Samples were electrophoresed on a 10% polyacrylamidegel and, following electrophoresis, the proteins were transferred tonitrocellulose membrane for Western blot analysis.

Probing the membrane with the core-specific monoclonal antibody JM122revealed a major single species in each sample which corresponds to coreprotein. The apparent molecular weights of the proteins made bypSFV.1-195 and two truncated variants, pSFV.1-173 and pSFV.1-169, areapproximately 21 kDa and are almost identical (FIG. 1A, lanes 1-3).Cleavage between the core and E1 coding regions occurs between residues191 and 192. However, there is additional data which reveals that thecore protein is further processed by cleavage around residue 174(Moradpour et al., 1996) this cleavage site will be referred to as theinternal processing site. The precise residue at which this secondcleavage event occurs is not known. Hence, in agreement with FIG. 1A,lanes 1-3, it would be predicted that the three constructs named abovewould generate products of similar molecular weights.

Additional evidence for a cleavage event close to residues 169-173occurring within tissue culture cells is shown in FIG. 1B. Here,polypeptides translated in vitro from the pGEM versions of 3 corevariants reveal that the unprocessed species made from pgHCV.1-195 islarger than that from pgHCV.1-173 (compare lanes 1 and 2). As would bepredicted from the coding sequences for the third truncated form ofcore, the major species synthesized from pSFV.1-153 has a lower apparentmolecular weight than that from pSFV.1-195 (FIG. 1A, compare lanes 1 and4). The major species made by the internal deletion mutantspSFV.Δ155-161 and pSFV.Δ161-166 are intermediate in size between thoseproduced by pSFV.1-195 and pSFV.1-153 (FIG. 1A, lanes 5 and 6). Again,this agrees well with predictions based on the number of amino acidsremoved in these variants (7 in pSFV.Δ155-161 and 6 in pSFV.Δ161-166).It is also evident that there is a significant amount of materialproduced by the two internal deletion variants which has a highermolecular weight than the fully processed form of core. This presumablyrepresents reduced cleavage at the internal processing site which mayresult from the removal of certain residues in these mutants which arenecessary for filly efficient processing. To conclude, the core proteinsand its variants produced by the SFV constructs can be detected by acore-specific antibody and their apparent molecular weights are inagreement with predictions from the nucleotide sequences and previouslypublished data.

Example 2 Intracellular Distribution of HCV Core Protein

Previous studies have revealed that the HCV core protein can associatewith lipid droplets within the cytoplasm of cells (Barba, G. et al.,1997; Moradpour, D. et al., 1996. This conclusion was arrived at bycombining the techniques of immune electron microscopy with the abilityto stain lipid with osmium tetroxide. However, this method suffers fromthe disadvantages that it is time-consuming and osmium tetroxide canstain other biological molecules (e.g proteins) in addition to lipid.Therefore, we developed a method for detecting proteins firstly byindirect immunofluorescence followed by staining of lipid droplets withthe oil-soluble colourant oil red O. Combined with the method ofconfocal microscopy, it is possible to visualize the intracellularlocalisations of core protein and lipid droplets separately andtogether. A typical example is shown in FIG. 2, panels A-C. Here, BHKC13 cells have been electroporated with pSFV.1-195 RNA and, followingincubation at 37° C. for 20 hours, the cells have been examined by bothindirect immunofluorescence and staining with oil red O. In panel A, thecore protein produced by pSFV.1-195 is seen to locate to vesicularstructures in the cytoplasm. Panel B reveals the distribution of lipiddroplets in the same cell. By merging these data (panel C), it isevident that core protein is sited around the lipid droplets. These datatherefore agree with previously published results for constructsexpressing the full-length coding region of core.

Example 3 Association of HCV Core Protein with Intracellular LipidDroplets Requires Amino Acids 161 to 166 and 125 to 144

Results with the constructs which produce truncated forms of coreprotein indicate that proteins consisting of 173 and 169 amino acids ofthe core coding region also locate to droplets (FIG. 2, panels D-I). Bycontrast, expressing only the N-terminal 153 residues results in loss oflocalisation to droplets and a diffuse cytoplasmic distribution isobserved (FIG. 2, panels J-L). Thus, residues of core protein betweenamino acids 154 and 169 are required for localisation to droplets.Studies with the internal deletion mutants pSFV.Δ155-161 andpSFV.Δ161-166 further examined segments within this 16 amino acid regionwhich may be important for core protein localisation. From the resultantdata, removal of residues between 155 and 161 did not affect lipiddroplet association whereas removal of residues between 161 and 166 gavea diffuse cytoplasmic pattern (FIG. 2, compare panels M-O with P-R).Hence, between residues 154 and 169, amino acids from 161 to 166 play anessential role in the ability of core protein to locate to lipiddroplets.

Further analysis of other internal deletion mutants (which removedresidues 9-43, 4-75 and 80-118) showed that the core proteins made bythese constructs continued to associate with lipid droplets (data notshown). Hence, these regions are dispensable for binding to droplets.However, a construct expressing a core variant in which residues 125 to144 had been deleted failed to distribute to droplets and gave a diffusecytoplasmic fluorescence (FIG. 2, panels S, T and U). This mutanttherefore identifies a second region in addition to the segment between161 and 166 which is necessary for association with droplets. The datasuggest that both sets of sequences are required for targeting to lipiddroplets. In agreement with these data, a core variant which istruncated at residue 152 and lacks amino acids 125 to 144 also fails tobind to droplets (FIG. 2, panels V, W and X). Additionally, thisprotein, in which both sets of targeting sequences are deleted, ispresent in low amounts in electroporated cells as a consequence ofdegradation.

Example 4 Effect of Localisation of Core Protein on the Lipid DropletAssociated Protein ADRP

At present, there are few proteins identified in mammalian cells whichare known to associate with lipid droplets. One protein which has beenrecently identified is ADRP which is ubiquitously expressed in a numberof tissue culture cell lines; ADRP mRNA has also been detected in arange of tissue types in mice. To examine whether the localisation ofcore to lipid droplets had any affect on ADRP, BHK C13 cells wereelectroporated with the series of pSFV constructs expressing coreprotein and its variants. An example of the data is shown in FIG. 3.Panels A to C show images of three cells following electroporation withpSFV.1-195, only one of which contains core protein (Panel B).Immunofluorescent results with the adipophilin antibody (panel A) revealthat ADRP is located on vesicular structures, consistent with itspreviously assigned association with lipid droplets. The protein isreadily detected in the cells which do not express core protein,however, it is considerably reduced in abundance in the core-expressingcell. Observations from this and a series of other experimentsconsistently revealed that cells expressing core protein from pSFV.1-195either lacked or contained barely detectable amounts of ADRP.

Nonetheless, some cells were also found in which both core protein andADRP were present; in general, such cells gave reduced fluorescence forthe core protein. Hence, it was concluded that the loss of ADRP wasrelated to the levels of expression of core in individual cells. Resultswith the variants of core which continue to locate to lipid dropletsgave identical data (see panels D-I and M-O). Thus, the majority ofcells expressing core protein from constructs pSFV.1-173, pSFV.1-169 andpSFV.Δ155-161 contained quantities of ADRP which were barely detectable.By contrast, ADRP continued to be readily found in cells producing coreproteins from pSFV.1-153 and pSFV.Δ161-166, the variants which do notassociate with lipid droplets (see panels J-L and P-R). Thus,association of core protein with lipid droplets correlates with a lossof ability to detect ADRP by immunofluorescence. Any cell typespecificity for this affect was tested by performing identicalexperiments in the rat hepatoma cell line, MCA RH7777. In these cells,core protein and its variants gave identical results for their abilityto associate with lipid droplets and this again correlated with thelevels of ADRP detected in core-expressing cells (FIG. 4). Thus theeffect of core protein on ADRP is not cell-type specific.

Example 5 The Ability of Core Protein to Associate with Lipid DropletsInduces a Loss of ADRP

The immunofluorescence data revealed that the association of coreprotein and its variants with lipid droplets led to an inability todetect ADRP. It was possible that this was due to masking of ADRP bycore. To examine directly the effect of core protein on the levels ofADRP, Western blot analysis was performed on cell extracts prepared atvarious times following electroporation with either pSFV.1-195 orpSFV.1-153 RNA. In parallel, immunofluorescence analysis was alsoperformed on these cells and this revealed that expression of the coreprotein produced by the two RNAs was apparent in greater than 90% ofcells.

Analysis with antibody JM122 indicated that core protein could bedetected from both constructs at 10 hours following electroporation andpeaked by about 20 hours (FIG. 5). The abundance of core proteinproduced by the two constructs was very similar by this time-point. Fromanalysis of these samples with the ADRP-specific antibody, it isapparent that there is no change in the abundance of ADRP followingelectroporation with the pSFV.1-153 RNA. A third set of cells in thisexperiment which was electroporated with SFV RNA which expresses the HCVE1 and E2 proteins also showed no reduction in ADRP levels with time. Bycontrast, there is a rapid reduction in ADRP levels to barely detectablequantities which mirrors the rise in core protein made from pSFV.1-195.

From staining of polyacrylamide gels with Coomassie brilliant blue,there were approximately equivalent amounts of protein in all samples.In addition, probing the membranes with another antibody for aendoplasmic reticulum-specific protein, calnexin, indicated that bothpSFV.1-195 and pSFV.1-153 samples had similar quantities of this proteinat the various times following electroporation. This affect of coreexpression on ADRP was consistently found in other experiments. Thus,the association of core protein with lipid droplets directly correlateswith a specific reduction in the abundance of this protein in cells.

Example 6 Targeting of a Protein of Interest Fused to an HCV TargetingSequence

To determine whether HCV core is capable of targeting a linked proteinof interest to intracellular lipid globules, a fusion construct composedof HCV core protein sequences linked to herpes simplex virus type 1(HSV-1) VP22 protein (encoded by gene UL49) is made.

The fusion construct comprises (from the N-terminus to the C-terminus)

1) The N-terminal 8 residues or the N-terminal 43 residues of HCV core.

2) Segments of the HSV-1 UL49 gene encoding residues 6-275 and 173-275.

3) An epitope tag consisting of residues ERKTPRVTGG. (McLauchlan, J. etal., 1994).

4) C-terminal residues of HCV core from 120-195 and 120-169.

It is also possible to use constructs wherein the N-terminal residues ofHCV and the C-terminal residues of HCV are contiguous and at theC-terminal or N-terminal end of the UL49 coding sequence (i.e. 2, 3, 1,4 or 1, 4, 2, 3 as denoted above).

These constructs will be expressed using the SFV vector system and theirability to associate with lipid droplets will be assessed byimmunofluorescence and oil red O-staining as described above. Theantibody used to detect the epitope tag is the anti-HCMV nuclear antigenantibody (MAb 9220) supplied by Capricorn Products. This antibody isalso used to verify by Western blot analysis that the fusion protein isof the predicted size and its abundance in comparison with coreconstructs.

Example 7 Identification of a Domain Required to Direct Core Protein ofHCV and GB Virus B to Lipid Droplets

In this example we have identified a conserved domain that is present inequivalent structural proteins encoded by HCV and GBV-B and directsthese proteins to lipid storage compartments.

GBV-B is a virus called GB virus-B. Information on GB virus-B has beenpresented by Beames et al (2000, Journal of Virology vol 74 No. 24, pp11764-11772) and Lanford et al (2001, Journal of Virology vol 75 No. 17,pp 8074-8081).

GBV-B was isolated from tamarins but the natural host of the virus isnot known. The GBV-B core protein is described in the example below. Thenucleic acid sequence of GBV-B is shown in Genbank Accession No.NC001655. GBV-B is the closest known related virus to HCV in terms ofsequence identity (29). GBV-B core protein is an example of a homologueof HCV core protein. An alignment of HCV and GVB-B amino acid sequenceis shown in FIG. 6. Furthermore, GVB-B is an example of a lipid globuletargeting sequence.

Lipid droplets are intracellular storage organelles that are found inall eukaryotic organisms and some prokaryotes (reviewed in 1-3). Theyconsist of a core of neutral lipid, comprising mainly triacylglycerolsand/or cholesterol esters, surrounded by a monolayer of phospholipids.Bounding the phospholipid layer is a proteinaceous coat. In mammaliancells, the principal lipid droplet binding proteins that have beenidentified are adipophilin (also called adipocytedifferentiation-related protein, ADRP; 7, 8) and a related family ofproteins termed the perilipins (9-11). Adipophilin is present in a widerange of cell types and in increased quantities in certain diseaseswhere intracellular lipid accumulation is evident (12-14). By contrast,expression of the perilipins is restricted to adipocytes andsteroidogenic cells (15, 16).

In addition to adipophilin and the perilipins, the core protein encodedby hepatitis C virus (HCV) also associates with lipid droplets inmammalian cells (17-19). HCV is the sole member of the hepacivirus genusthat is incorporated into the Flaviviridae family along with two othergenera, the flavi- and pestiviruses. All of the Flaviviridae arepositive-sense, single stranded RNA viruses that have similar genomearrangements and share sequence similarities. HCV core is a structuralcomponent of the virus particle and, by analogy with flavi- andpestiviruses, it is likely to be the sole component of the capsid (20,21). The protein is generated from a polyprotein encoded by the viralgenome by cleavage at the endoplasmic reticulum (ER; 22-26). Expressionof core can lead to the genesis of lipid droplets in tissue culturecells (18) and the development of steatosis in transgenic mice (27).Moreover, interaction between core and apolipoprotein AII, a componentof lipid droplets, has been demonstrated (28). These data indicate that,not only does core associate with lipid droplets, but it also may havethe capacity to influence metabolic events within the cell involving thestorage of lipid.

To understand the significance of the interaction between HCV core andlipid droplets, a region within the viral protein that is essential forits association with these storage organelles had been identified (19).In this example, studies have been carried out to determine whetherthere is any similarity between the sequences within this region andthose of GBV-B, which has significant sequence identity to HCV although,as yet, it remains unclassified among the genera of the Flaviviridae(29). GBV-B shares a tropism for liver hepatocytes with HCV and isinfectious in tamarins (30, 31). Thus, it has been suggested that GBV-Binfection of tamarins may be a surrogate model system for HCV infectionof humans (31). However, few comparative studies between equivalentproteins encoded by the two viruses have been conducted.

TABLE 1 Oligonucleotides used to generate constructs. Nucleotide NameOligonucleotide Sequence Position^(a) HCV CAT GGG GTA CAT AGC GCT CGTCGG 1 CGC CGC CTT AAG AGG CGC TGC GAG GGC C HCV CTA GAG AGC GCA AGA CGCCCC GCG 2 TCA CCG GCG GCG GBV GGA GAT CTC GTA GAC CGT AGC ACA 428-448 B1TG GBV GGG GAT CCC TAG TGG ACA CCG AAC 842-868 B2 CAA CCA GTA GCC CA GBVGGG GAT CCT CAG ATC ACA CAA CCA 1003-1029 B3 GGC TCG TGT AGG GBV GGG TACTCT AGA GTG ATA GGC CTG 1618-1639 B4 GTC GBV CTA GAG AGC GCA AGA CGC CGCGGG B5 TCA CCG GTG GCT CTC GCA ATC TTG G ^(a)Nucleotide positions on theGBV-B viral genome (Genbank Accession No. NC001655) for the GBV-Bprimers

Materials and Methods Construction of Plasmids

Construction of plasmids pSFV/1-195 and pSFV/1-169 has been describedpreviously (19). The codons for the proline residues in these constructswere mutated to encode alanine by insertion of an oligonucleotide (HCV1in Table 1). This oligonucleotide was inserted between BspHI and BstX1sites in construct pgHCV/□135-144 (19) to give plasmid pgHCV/1-195_((P)_(A)); the BspHI site is not a natural site in the sequence for HCVstrain Glasgow and was introduced during the construction ofpgHCV/□135-144. To generate pSFV/1-195_((P) _(A)) and pSFV/1-169_((P)_(A)), a 502 bp fragment from pgHCV/1-195_((P) _(A)), produced bycleavage by BglII and BstEII, was inserted into pSFV/1-195 andpSFV/1-169 digested with the same enzymes. A tagged version of HCV corewas generated by firstly converting the nucleotide sequence in pg/1-195that encodes amino acids 116 and 117 from TCG CGC to TCT AGA; thisintroduced a novel XbaI site into the HCV core-coding region withoutchanging the encoded amino acids. An oligonucleotide (HCV2 in Table 1)that encoded the epitope tag (32) was introduced into the XbaI site togive plasmid pg/1-195tag. The tagged version of core was transferred asa BglII fragment from pg/1-195tag into Semliki Forest virus (SFV)expression vector pSFV1 (33) to give construct pSFV/1-195tag.

To express portions of the GBV-B polyprotein, relevant regions wereamplified by PCR from a construct pGBB (30). pGBB contains the consensussequence for an infectious molecular clone of GBV-B (30). Primers forPCR amplification were derived from the viral sequences in pGBB and wereused in the following pairs to produce DNA fragments that encodedN-terminal regions of the GBV-B polyprotein: residues 1-141, GBV-Bprimers 1 and 2; residues 1-194, GBV-B primers 1 and 3; residues 1-398,GBV-B primers 1 and 4. Amplified fragments were introduced initiallyinto plasmid pGEM1 and thereafter into pSFV1 by standard cloningtechniques. To permit detection of GBV-B core, an epitope tag (32) wasintroduced into the coding region immediately following amino acidresidue 85 (FIG. 6). This was accomplished by firstly introducing anovel XbaI site at nucleotide residue 695 by converting the sequencefrom TCT CGC to TCT AGA; this did not alter the encoded amino acidsequence. An oligonucleotide encoding the epitope tag (GBV-B5 inTable 1) was inserted between this XbaI site and a TfiI site (position708 in the native GBV-B nucleotide sequence). The final SFV constructsthat contained tagged versions of GBV-B core were termed pSFV/GB 1-141,pSFV/GB1-194 and pSFV/GB 1-398.

Maintenance of Tissue Culture Cells and Treatment with MG132

Baby hamster kidney (BHK) C13 cells were grown and maintained in Glasgowminimal Eagle's Medium supplemented with 10% new-born calf serum (CS),4% tryptose phosphate broth, and 100 IU/ml penicillin/streptomycin(ETC10). To treat 1BHK cells with MG132 (supplied by Boston Biochem),cells were incubated for 5 h after electroporation at 37° C. and themedia was replaced with fresh media containing the protease inhibitor ata final concentration of 2.5 μg/ml. Incubation was continued at 37° C.for a further 12 h before the cells were either harvested for Westernblot analysis or fixed for indirect immunofluorescence studies.

Immunological Reagents

The monoclonal antibodies (MAb) used to detect HCV core protein (MAbJM122) and the epitope tag (MAb 9220) have been described previously (19and 32 respectively).

1. In Vitro Transcription and Electroporation of SFV RNA into Cells

RNA was transcribed in vitro from recombinant pSFV constructs linearizedwith SpeI. BHK cells were electroporated with in vitro transcribed RNAas described previously (19, 36). Cells were incubated at 37° C. for 15h and then harvested.

Preparation of Cell Extracts, Polyacrylamide Gel Electrophoresis andWestern Blot Analysis

Extracts were prepared and polyacrylamide gel electrophoresis performedas described previously (19, 36).

For Western blot analysis, proteins separated on polyacrylamide gelswere transferred to nitrocellulose membrane. After blocking with 3%gelatin, 4 mM Tris-HCl, pH 7.4, 100 mM NaCl, membranes were incubatedwith antibodies (diluted to 1/500) in 1% gelatin, 4 mM Tris-HCl, pH 7.4,100 mM NaCl, 0.05% Tween 20. After washing, bound antibody was detectedusing a horseradish peroxidase-conjugated secondary antibody followed byenhanced chemiluminescence (Amersham, UK).

Indirect Immunofluorescence and Staining of Lipids

Cells on 13 mm coverslips were fixed for 30 min in 4% paraformaldehyde,0.1% Triton X-100 (prepared in phosphate-buffered saline) at 4° C.Following washing with phosphate-buffered saline (PBS) and blocking withPBS/CS (PBS containing 1% newborn calf serum), cells were incubated withprimary antibody (diluted in PBS/CS at 1/200 for JM122 and 9220antibodies) for 2 h at room temperature. Cells were washed extensivelywith PBS/CS and then incubated with conjugated secondary antibody(either anti-mouse or anti-rabbit IgG raised in goat) for 2 h at roomtemperature. Cells were washed extensively in solutions of PBS/CSfollowed by PBS. Staining of lipid droplets by oil red O was performedas described previously (19). Cells were rinsed finally with H₂O beforemounting on slides using Citifluor (Citifluor Ltd., UK). Samples wereanalyzed using a Zeiss LSM confocal microscope.

Computing

Sequences were aligned using the CLUSTAL W alignment program (37) andhydropathicity plots generated by ProtScale (38).

Results

A Domain in the HCV Core Protein that is Absent in Flavi- andPestiviruses but Present in GBV-B

Previously, it was proposed that the HCV core protein consisted of 3domains and that the second of these domains was absent in relatedpesti- and flaviviruses (19, 39). Although there is a lack of sequenceidentity between viral sequences in the Flaviviridae, each of the capsidproteins in members of this virus family have a high content of basicamino acids (40). Therefore, the comparisons described here were basedon the proportion of positively charged residues in predicted codingregions accompanied by hydropathicity plot studies. It was found thatthe mature capsid (C) protein of yellow fever virus (YF), a flavivirus,had a high proportion of basic residues (27%). In contrast, the signalpeptide sequence that is removed from C protein upon maturation did notcontain any basic amino acids. Up to amino acid 117 of the HCV coreprotein, 23% of residues were basic and this dropped to 7% betweenresidues 118-173. Thus, the N-terminal 117 amino acids of HCV core havea similar character to those in the YF C protein but there are nosequences corresponding to the region between 118 and 173 of the HCVpolypeptide. Analysis of the hydropathicity of the HCV core protein alsorevealed a highly hydrophilic region containing a similar segment ofbasic residues (RRRSR) between residues 113-117 (FIG. 6). The regionbetween residues 118 and 173 is referred to as domain 2 (19, 39).

A closely related virus to HCV is GBV-B, a virus that was isolated fromtamarins but whose natural host is not known. To date, the proteolyticevents to generate the mature proteins of GBV-B have been assumed fromcomparison with HCV polypeptide processing (29). Comparing the putativeGBV-B core sequence with that of HCV did not identify any stretches ofsimilarity until residue 75 of GBV-B (FIG. 6). According to the sequencealignment, domain 1 for GBV-B core ended at residue 85 (corresponding toresidue 117 in HCV core; FIG. 6) and thus, for this domain, the GBV-Bsequence was shorter than that for HCV. The overall sequence identity(sequence homology) between these domains in HCV and GBV-B was about22%. From residue 86 and up to residue 140 of GBV-B, sequence identitybetween the two viral sequences increased to 41% and apart from oneadditional residue in GBV-B, the sequences were colinear (FIG. 6). InHCV core, this segment is composed principally of domain 2 and indicatedthat sequences corresponding to this region are present in GBV-B.Sequence identity between the residues 140-156 for GBV-B and 174-191 forHCV (corresponding to the signal peptide sequences) was slightly lowerat 38% and reduced further in the equivalent E1 sequences (approx. 25%).Distribution of basic residues in the putative GBV-B core proteinrevealed a high lysine/arginine content (21%) in the N-terminal regionup to amino acid 85, a reduced percentage beyond this point (3.6%between amino acids 86-136) and no positively charged amino acids in thesignal peptide sequence. This pattern of distribution corresponded tothat present in HCV core. From these data, it was concluded that theputative GBV-B core protein shares the same domain arrangement as itscounterpart in HCV.

Intracellular Localization of the GBV-B Core Protein

Previous studies on the intracellular localization of HCV core showedthat the protein was directed to lipid droplets and the primary sequencedeterminants for this localization were present in domain 2 (19). Sincethe region of highest homology between the core proteins of GBV-B andHCV encompassed this domain, we expressed a N-terminal region of theGBV-B polyprotein using pSFV/GB1-398 in which the coding region extendedbeyond the predicted C-terminus of E1 to analyze the intracellularlocalization of GBV-B core. As immunological reagents against GBV-Bproteins were not available, we placed a short epitope tag (32) atresidue 85 to detect the protein. Placing this tag into thecorresponding region of the HCV core protein did not affect itsintracellular localization and the protein was present on lipiddroplets. Indirect immunofluorescence of cells electroporated with RNAfrom pSFV/GB1-398 using an antibody, 9220, that recognizes the epitopetag revealed staining around lipid droplets stained with oil red O.Western blot analysis of cell extracts indicated that the size of coreprotein detected was approximately 17 kDa. This is consistent with aproduct of about 155 amino acids (comprising 143 amino acids of GBV-Band 12 amino acids of the epitope tag).

To further verify the intracellular localization of GBV-B core and thelikely events involved in its maturation, two other constructscontaining GBV-B sequences were examined. Firstly, a constructpSFV/GB1-141 that would correspond approximately in size to the productdetected with pSFV/GB1-398. Analysis of cells expressing the taggedproduct produced by pSFV/GB1-141 again revealed localization of theprotein around lipid droplets. The size of the protein made bypSFV/GB1-141 was only slightly smaller than that made by pSFV/GB1-398.Another construct that expressed the N-terminal 194 residues of GBV-Bgave a major product that was identical in size to that produced bypSFV/GB1-398; a second minor band of about 20 kDa represented uncleavedprotein. It was concluded that, in common with HCV core, the equivalentGBV-B protein is directed to lipid droplets in tissue culture cells andcleavage of the GBV-B polyprotein to produce the mature form of core isdirected by cellular peptidases. Our studies on HCV core revealed thatdomain 2 contained the sequences essential for lipid dropletassociation. Based on our sequence comparisons, this domain is presentalso in the equivalent GBV-B protein. Hence, we propose that theessential sequences for association with lipid droplets reside withinthe corresponding region of GBV-B core.

Discussion

From previous analysis and data presented in this example, it wasproposed that the HCV core protein consisted of three domains (19, 39).Domain 1 corresponded to the mature core protein of flaviviruses whileno sequences equivalent to domain 2 were present in either pesti- orflaviviruses. Domain 3 contained the signal peptide sequence thatdirects the HCV E1 glycoprotein to the ER lumen. Here, the sequencecomparisons were extended to include GBV-B, a virus that is the closestknown related virus to HCV in terms of sequence identity (29). Fromcomparisons of the two viral sequences, domain 2 in HCV core was foundalso in the corresponding GBV-B protein. Sequence identity between thepredicted amino acid sequences of HCV and GBV-B in the region containingdomain 2 was higher (approx. 41%) than that in the N- and C-terminalflanking regions. This degree of similarity would imply that thesedomains in HCV and GBV-B might perform similar functions. Extensivemutational analysis and immunolocalization studies of the HCV coreprotein showed that domain 2 contained the key elements for directingthe protein to lipid droplets (19). The data presented in this exampleindicated that the core protein of GBV-B also could associate with theselipid storage structures. Thus, we conclude that a function of thesedomains is to direct the core protein of the two viruses to lipidstorage organelles.

From the above evidence, it was concluded that the HCV and GBV-B coreproteins have similar domain configurations. Based on our data (19 andthis example), domain 2 in both proteins represented sequences thatdirected them to lipid droplets. Comparison with mammalian proteins thatassociate with these structures did not identify any region in suchproteins with features similar to those in domain 2.

The HCV and GBV-B core proteins each contained two closely spacedproline residues within domain 2. Substitution of these prolines in HCVcore abolished lipid droplet association. We have shown previously thatremoval of short sequence elements of domain 2 from HCV core preventedlipid droplet association (19). Based on these findings, we suggest thatthe HCV and GBV-B core may be members of a family of proteins that sharesimilar sequence characteristics for targeting to lipid droplets.

To summarize this example, in mammalian tissue culture cells, the coreprotein of hepatitis C virus (HCV) is located at the surface of lipiddroplets, which are cytoplasmic structures that store lipid. Thecritical amino acid sequences necessary for this localization are in aregion of core protein that is absent in flavi- and pestiviruses, whichare related to HCV. From sequence comparisons, this region in HCV corewas present in the corresponding protein of GB virus-B (GBV-B), anothervirus whose genomic sequence has significant similarity (homology asdefined herein) to HCV. Expression of the putative GBV-B core proteinrevealed that it also was directed to lipid droplets. Extending thecomparisons to mammalian cellular proteins, there were no amino acidsimilarities with the domains for lipid droplet association in HCV core.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

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18 1 630 DNA Hepatitis C Virus CDS (43)...(630) 1 ggtgcttgcg agtgccccgggaggtctcgt agaccgtgca cc atg agc acg aat 54 Met Ser Thr Asn 1 cct aaacct caa aga aaa acc aaa cgt aac acc aac cgt cgc cca cag 102 Pro Lys ProGln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 5 10 15 20 gac gttaag ttc ccg ggt ggc ggt cag atc gtt ggt gga gtt tac ttg 150 Asp Val LysPhe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 25 30 35 ttg ccg cgcagg ggc cct aga ttg ggt gtg cgc gcg acg agg aag act 198 Leu Pro Arg ArgGly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr 40 45 50 tcc gag cgg tcgcaa cct cga ggt aga cgt cag cct atc ccc aag gca 246 Ser Glu Arg Ser GlnPro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala 55 60 65 cgt cgg ccc aag ggcagg aac tgg gct cag ccc ggg tat cct tgg ccc 294 Arg Arg Pro Lys Gly ArgAsn Trp Ala Gln Pro Gly Tyr Pro Trp Pro 70 75 80 ctc tat ggc aat gag ggttgc ggg tgg gcg gga tgg ctc ctg tcc ccc 342 Leu Tyr Gly Asn Glu Gly CysGly Trp Ala Gly Trp Leu Leu Ser Pro 85 90 95 100 agt ggc tct cgg cct agttgg ggc ccc aac gac ccc cga cgt agg tcg 390 Ser Gly Ser Arg Pro Ser TrpGly Pro Asn Asp Pro Arg Arg Arg Ser 105 110 115 cgc aat ttg ggt aag gtcatc gat acc ctt acg tgc ggc ttc gtc gat 438 Arg Asn Leu Gly Lys Val IleAsp Thr Leu Thr Cys Gly Phe Val Asp 120 125 130 ctc atg ggg tac ata ccgctc gtc ggc gcc cct ctt aga ggc gct gcc 486 Leu Met Gly Tyr Ile Pro LeuVal Gly Ala Pro Leu Arg Gly Ala Ala 135 140 145 agg gcc ctg gcg cat ggcgtc cgg gtt ctg gaa gac ggt gtg aac tat 534 Arg Ala Leu Ala His Gly ValArg Val Leu Glu Asp Gly Val Asn Tyr 150 155 160 gca aca ggt aac ctt cctggt tgc tct ttc tct atc ttc ctt ctg gcc 582 Ala Thr Gly Asn Leu Pro GlyCys Ser Phe Ser Ile Phe Leu Leu Ala 165 170 175 180 ctg ctc tct tgc ctgact gtg ccc gct tca gcc tac caa gtg cgc aac 630 Leu Leu Ser Cys Leu ThrVal Pro Ala Ser Ala Tyr Gln Val Arg Asn 185 190 195 2 60 DNA Hepatitis CVirus CDS (1)...(60) Corresponds to amino acids 125-144 of the HCV Coreprotein sequence 2 acc ctt acg tgc ggc ttc gtc gat ctc atg ggg tac ataccg ctc gtc 48 Thr Leu Thr Cys Gly Phe Val Asp Leu Met Gly Tyr Ile ProLeu Val 1 5 10 15 ggc gcc cct ctt 60 Gly Ala Pro Leu 20 3 18 DNAHepatitis C Virus CDS (1)...(18) Corresponds to Hepatitis C Virus coreprotein amino acids 161-166 3 ggt gtg aac tat gca aca 18 Gly Val Asn TyrAla Thr 1 5 4 31 PRT Artificial Sequence A branched peptide containingresidues 5-27 or the core protein ecoded by HCV strain Glasgow. Sites 1and 12 are degenerate with site 1 being Ala or Pro and site 12 being Ileor Asn 4 Ala Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Ile Arg Arg Pro Gln1 5 10 15 Asp Val Lys Phe Pro Gly Gly Lys Lys Lys Lys Lys Lys Lys Ala 2025 30 5 11 DNA Artificial Sequence Oligonucleotide used to produce theHepatitis C virus core deletion plasmids 5 gctgagatct a 11 6 29 DNAArtificial Sequence Oligonucleotide used to produce the Hepatitis Cvirus core deletion plasmids 6 gtaaccttcc tggttgctct tgagatcta 29 7 17DNA Artificial Sequence Oligonucleotide used to produce the Hepatitis Cvirus core deletion plasmids 7 gtaacctttg agatcta 17 8 18 DNA ArtificialSequence Oligonucleotide used to produce the Hepatitis C virus coredeletion plasmids 8 ctggcgcatt gagatcta 18 9 28 DNA Artificial SequenceOligonucleotide used to produce the Hepatitis C virus core deletionplasmids 9 ctggcccatg gtgttaacta tgcaacag 28 10 31 DNA ArtificialSequence Oligonucleotide used to produce the Hepatitis C virus coredeletion plasmids 10 ctggcccatg gcgtccgggt tctggaagac g 31 11 32 DNAArtificial Sequence Oligonucleotide used to produce the Hepatitis Cvirus core deletion plasmids 11 gaggcgctgc cagggccctg gcgtgagatc ta 3212 52 DNA Artificial Sequence Oligonucleotide used to generateconstructs, identified as HCV1 12 catggggtac atagcgctcg tcggcgccgccttaagaggc gctgcgaggg cc 52 13 36 DNA Artificial SequenceOligonucleotide used to generate constructs, identified as HCV2 13ctagagagcg caagacgccc cgcgtcaccg gcggcg 36 14 26 DNA Artificial SequenceOligonucleotide used to generate constructs, identified as GBV-B1 14ggagatctcg tagaccgtag cacatg 26 15 38 DNA Artificial SequenceOligonucleotide used to generate constructs, identified as GBV-B2 15ggggatccct agtggacacc gaaccaacca gtagccca 38 16 36 DNA ArtificialSequence Oligonucleotide used to generate constructs, identified asGBV-B3 16 ggggatcctc agatcacaca accaggctcg tgtagg 36 17 27 DNAArtificial Sequence Oligonucleotide used to generate constructs,identified as GBV-B4 17 gggtactcta gagtgatagg cctggtc 27 18 49 DNAArtificial Sequence Oligonucleotide used to generate constructs,identified as GBV-B5 18 ctagagagcg caagacgccg cgggtcaccg gtggctctcgcaatcttgg 49

What is claimed is:
 1. A polynucleotide encoding a fusion protein,wherein said fusion protein comprises a lipid globule targeting sequencelinked to a protein of interest (POI), wherein the targeting sequencecomprises a hepatitis C virus (HCV) core protein or fragment orhomologue of a hepatitis C virus (HCV) core protein.
 2. A polynucleotideaccording to claim 1 wherein the fragment comprises amino acids from 125to 144 and/or 161 to 166 of the HCV core protein together with ahydrophilic sequence.
 3. A polynucleotide according to claim 1 whereinthe POI comprises at least one epitope.
 4. A polynucleotide according toclaim 3 wherein the POI is a viral or bacterial protein or fragmentthereof.
 5. A polynucleotide according to claim 4 wherein the POI is anHCV antigen.
 6. A polynucleotide according to claim 1 operably linked toa control sequence permitting expression of the protein in a suitablehost cell.
 7. A polynucleotide according to claim 6 wherein the hostcell is an adipocyte.
 8. A polynucleotide according to claim 7 whereinthe host cell is a milk-secreting cell.
 9. A polynucleotide according toclaim 8 wherein said targeting sequence comprises a hepatitis C virus(HCV) core protein homologue of a fragment thereof, wherein saidhomologue is GB virus-B core protein or a fragment thereof.
 10. Anucleic acid vector comprising a polynucleotide according to claim 1.11. A nucleic acid vector according to claim 10 wherein saidpolynucleotide is operably linked to a polylinker cloning site.
 12. Ahost cell comprising a polynucleotide according to claim
 1. 13. A hostcell comprising a nucleic acid vector according to claim
 10. 14. Amethod comprising expressing a polynucleotide according to claim
 1. 15.A method according to claim 14 wherein the expressed product isisolated.
 16. A fusion protein comprising a lipid globule targetingsequence linked to a protein of interest (POI) wherein the targetingsequence comprises a hepatitis C virus (HCV) core protein or homologueor fragment of a hepatitis C virus (HCV) core protein or a fragment ofsaid hepatitis C virus (HCV) core protein homologue.